S100s are a unique group of proteins containing the EF-hand calcium binding motif. They are characterized by cell-type and cell cycle specific expression, as well as deregulated expression in some neurological and inflammatory disorders and certain cancers. On the structural level, Sl00s are distinguished from other EF-hand proteins by their unique architecture. To date, structural biology studies on these proteins have provided detailed information on the S100 structure and the response to binding calcium, but little insight into how these proteins affect signaling within a larger cellular context. This proposal is aimed at understanding how S100 proteins modulate the activities of their binding partners and participate in signal transduction pathways, by studying one intracellular and one extracellular signaling system. A multi-disciplinary strategy, that incorporates biochemical and structural approaches, will be utilized. The broad, long-term objective of this research program is to determine the structural basis for the cellular activities of the S100 proteins, so that their roles in health and disease may be better understood. S100A6 has been shown to interact physically and functionally with Siah-1 interacting protein (SIP), which Is an essential component of an E3 ligase complex that ubiquitinates beta-catenin as part of the regulation of cell proliferation during embryogenesis. In Aim 1, we propose to: (i) define the structural basis for the interaction of S100A6 and SIP; (ii) determine the structures and the structural organization of SlP's three Idomains; (iii) examine how S100 protein binding affects the organization of SIP domains and its interactions with other SIP binding proteins. Several S100 proteins interact with RAGE, a receptor for Advanced Glycation Endproducts (AGEs). AGE binding to RAGE has been linked with the progression of atherosclerosis in diabetes. In Aim 2, we propose to: (i) define the structural basis for the interaction of RAGE with S100 proteins; (ii) determine how binding of S100A6 affects the structural organization of the extracellular domains of RAGE; (iii) characterize the cross-talk between binding of S100 proteins and AGEs. These results will provide the insights on the structural basis of S100 function in AGE receptor signaling, the first such information on function of S100 proteins in the extracellular milieu.